Elevated levels of plasma very low-density lipoproteins (VLDLs) multiply the risk of atherosclerosis and coronary artery diseases, which currently poses an egregious impact to public health in the United States. VLDLs are synthesized in the liver and secreted into the blood. The rate-determining step in the secretion of VLDLs from the liver is their transport from their site of biogenesis, the endoplasmic reticulum (ER), to the Golgi. This step is physiologically regulatable and represents a potential therapeutic target in controlling elevated concentrations of plasma VLDLs. We propose to study how nascent VLDL exits from the ER and gets delivered to the Golgi at the molecular level. Our laboratory has shown that VLDL exits the ER in a specialized vesicle, the VLDL transport vesicle (VTV) which is different from other ER-derived vesicles e.g. protein transport vesicle (PTV) and pre-chylomicron transport vesicle (PCTV) in its size, buoyant density and protein composition. We reported earlier the VTV proteome, which revealed that reticulon-3A2 (RTN3A2) and small Valosine Containing Peptide (VCP)-interacting protein (SVIP) are uniquely present in VTV. Our preliminary studies show that apolipoproteinB100 (apoB100), a VLDL structural protein, plays a key role in VLDL- packaging into the VTV. Interestingly, we found that RTN3A2 selectively interacts with VLDL-apoB100 and is required for VLDL-exit but not for albumin-exit from the same hepatic ER. The first specific aim of this proposal is to identify cargo-selecting binding sites on RTN3A2 and cargo-selective binding sites on apoB100 for VLDL inclusion in VTV. In preliminary studies, we have identified another small Mr protein, SVIP, which is concentrated in VTVs as compared with their parent hepatic ER membranes. Our results show that SVIP interacts specifically with apoB100, the primary VTV-cargo protein and Sar1 but not with albumin, a PTV-cargo protein suggesting a role for SVIP in VTV-mediated transport of the VLDL. Our second specific aim will be to establish the role of SVIP in VTV-biogenesis and the ER-exit of apoB100 in primary hepatocytes. We found in our preliminary studies that the formation of VTV-Golgi fusion-competent SNARE-complex requires cytosolic proteins. In preliminary studies, we identified a functionally active cytosolic fraction that suppots SNARE- complex formation. SDS-PAGE analysis revealed that the active fraction contains only 3 proteins. We have identified two of these proteins. In specific aim three of this grant, we propose to identify the third protein and test the functional roles of these proteins in VTV-Golgi fusion-complex assembly.